RESUMO
The discovery of genetic variants in the cholinergic receptor nicotinic CHRNA5-CHRNA3-CHRNB4 gene cluster associated with heavy smoking and higher relapse risk has led to the identification of the midbrain habenula-interpeduncular axis as a critical relay circuit in the control of nicotine dependence. Although clear roles for α3, ß4, and α5 receptors in nicotine aversion and withdrawal have been established, the cellular and molecular mechanisms that participate in signaling nicotine use and contribute to relapse have not been identified. Here, using translating ribosome affinity purification (TRAP) profiling, electrophysiology, and behavior, we demonstrate that cholinergic neurons, but not peptidergic neurons, of the medial habenula (MHb) display spontaneous tonic firing of 2-10 Hz generated by hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels and that infusion of the HCN pacemaker antagonist ZD7288 in the habenula precipitates somatic and affective signs of withdrawal. Further, we show that a strong, α3ß4-dependent increase in firing frequency is observed in these pacemaker neurons upon acute exposure to nicotine. No change in the basal or nicotine-induced firing was observed in cholinergic MHb neurons from mice chronically treated with nicotine. We observe, however, that, during withdrawal, reexposure to nicotine doubles the frequency of pacemaking activity in these neurons. These findings demonstrate that the pacemaking mechanism of cholinergic MHb neurons controls withdrawal, suggesting that the heightened nicotine sensitivity of these neurons during withdrawal may contribute to smoking relapse.
Assuntos
Relógios Biológicos/efeitos dos fármacos , Neurônios Colinérgicos , Habenula , Nicotina/efeitos adversos , Agonistas Nicotínicos/efeitos adversos , Síndrome de Abstinência a Substâncias , Animais , Cardiotônicos/farmacologia , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/patologia , Habenula/metabolismo , Habenula/patologia , Habenula/fisiopatologia , Humanos , Camundongos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Pirimidinas/farmacologia , Fumar/metabolismo , Fumar/patologia , Fumar/fisiopatologia , Abandono do Hábito de Fumar , Síndrome de Abstinência a Substâncias/metabolismo , Síndrome de Abstinência a Substâncias/patologia , Síndrome de Abstinência a Substâncias/fisiopatologiaRESUMO
Alpha-Synuclein (α-Syn) aggregation is a pathological feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease (PD). Here, we explored the efficacy of N,N,N',N'-tetraethyl-10H-phenothiazine-3,7-diamine dihydrochloride (LETC), a protein aggregation inhibitor, on α-Syn aggregation. In both cellular models and transgenic mice, α-Syn aggregation was achieved by the overexpression of full-length human α-Syn fused with a signal sequence peptide. α-Syn accumulated in transfected DH60.21 neuroblastoma cells and α-Syn aggregation was inhibited by LETC with an EC50 of 0.066 ± 0.047 µM. Full-length human α-Syn overexpressing Line 62 (L62) mice accumulated neuronal α-Syn that was associated with a decreased motor performance in the open field and automated home cage. LETC, administered orally for 6 weeks at 10 mg/kg significantly decreased α-Syn-positive neurons in multiple brain regions and this resulted in a rescue of movement deficits in the open field in these mice. LETC however, did not improve activity deficits of L62 mice in the home cage environment. The results suggest that LETC may provide a potential disease modification therapy in synucleinopathies through the inhibition of α-Syn aggregation.
Assuntos
Doença de Parkinson , Sinucleinopatias , Camundongos , Humanos , Animais , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Sinucleinopatias/patologia , Doença de Parkinson/metabolismo , Camundongos Transgênicos , Encéfalo/metabolismoRESUMO
At synaptic terminals, high voltage-activated Ca(v)2.1 and Ca(v)2.2 calcium channels have an essential and joint role in coupling the presynaptic action potential to neurotransmitter release. Here we show that membrane-tethered toxins allowed cell-autonomous blockade of each channel individually or simultaneously in mouse neurons in vivo. We report optimized constitutive, inducible and Cre recombinase-dependent lentiviral vectors encoding fluorescent recombinant toxins, and we also validated the toxin-based strategy in a transgenic mouse model. Toxins delivered by lentiviral vectors selectively inhibited the dopaminergic nigrostriatal pathway, and transgenic mice with targeted expression in nociceptive peripheral neurons displayed long-lasting suppression of chronic pain. Optimized tethered toxins are tools for cell-specific and temporal manipulation of ion channel-mediated activities in vivo, including blockade of neurotransmitter release.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Transmissão Sináptica/efeitos dos fármacos , ômega-Conotoxinas/farmacologia , Animais , Canais de Cálcio Tipo N/efeitos dos fármacos , Células Cultivadas , Dopamina/metabolismo , Humanos , Integrases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dor/prevenção & controle , Ratos , Ratos Wistar , ômega-Conotoxinas/metabolismoRESUMO
The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors. Multiple clinical trials have been undertaken for this target in the past 15 years; however, we are still to see market approval of the first liver-targeted adeno-associated virus (AAV)-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically and clinically predictive preclinical models. To this end, this study reports findings of a functional evaluation of 6 AAV vectors in 12 preclinical models of the human liver, with the aim to uncover which combination of models is the most relevant for the identification of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver.
Assuntos
Dependovirus , Fígado , Humanos , Dependovirus/genética , Fígado/metabolismo , Terapia Genética/métodos , Hepatócitos/metabolismo , Proteínas do Capsídeo/metabolismo , Tropismo , Vetores Genéticos/genéticaRESUMO
The accumulation of alpha-synuclein (α-Syn) into Lewy bodies in cortical and subcortical regions has been linked to the pathogenesis of synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). While there is a strong link between synuclein aggregates and the reduction in dopamine function in the emergence of PD, less is known about the consequences of α-Syn accumulation in glutamatergic neurons and how this could be exploited as a therapeutic target. Transgenic h-α-synL62 (L62) mice, in which synuclein aggregation is achieved through the expression of full-length human α-Syn fused with a signal sequence peptide, were used to characterise glutamatergic transmission using a combination of behavioural, immunoblotting, and histopathological approaches. The protein aggregation inhibitor hydromethylthionine mesylate (HMTM) alone, or in combination with the glutamatergic compounds 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine hydrochloride (MTEP) and memantine, was used to target α-Syn aggregation. We show that accumulation of α-Syn aggregates in glutamatergic synapses affected synaptic protein expression including metabotropic glutamate receptor 5 (mGLUR5) levels and ratio of N-methyl-d-aspartate (NMDA) receptor subunits GluN1/GluN2A. The ratio of NMDA receptor subunits and levels of mGLUR5 were both normalised by HMTM in L62 mice. These alterations, however, did not affect glutamate release in synaptosomes derived from L62 mice or behavioural endpoints following pharmacological manipulations of glutamate functions. Our results confirm that HMTM acts in the L62 mouse model of PD as an inhibitor of pathological aggregation of synuclein and show that HMTM treatment normalises both the ratio of NMDA receptor subunits and mGLUR5 levels. These findings support the potential utility of HMTM as a disease-modifying treatment for PD aiming to reduce synuclein aggregation pathology.
Assuntos
Doença de Parkinson , Sinucleinopatias , Animais , Glutamatos/uso terapêutico , Humanos , Azul de Metileno/análogos & derivados , Camundongos , Camundongos Transgênicos , Doença de Parkinson/tratamento farmacológico , Receptores de N-Metil-D-Aspartato/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Less than 80 Sumatran rhinos (SR, Dicerorhinus sumatrensis) are left on earth. Habitat loss and limited breeding possibilities are the greatest threats to the species and lead to a continuous population decline. To stop the erosion of genetic diversity, reintroduction of genetic material is indispensable. However, as the propagation rate of captive breeding is far too low, innovative technologies have to be developed. Induced pluripotent stem cells (iPSCs) are a powerful tool to fight extinction. They give rise to each cell within the body including gametes and provide a unique modality to preserve genetic material across time. Additionally, they enable studying species-specific developmental processes. Here, we generate iPSCs from the last male Malaysian SR Kertam, who died in 2019, and characterize them comprehensively. Differentiation in cells of the three germ layers and cerebral organoids demonstrate their high quality and great potential for supporting the rescue of this critically endangered species.
RESUMO
Understanding information flow in sensory pathways requires cell-selective approaches to manipulate the activity of defined neurones. Primary afferent nociceptors, which detect painful stimuli, are enriched in specific voltage-gated sodium channel (VGSC) subtypes. Toxins derived from venomous animals can be used to dissect the contributions of particular ion currents to cell physiology. Here we have used a transgenic approach to target a membrane-tethered isoform of the conotoxin MrVIa (t-MrVIa) only to nociceptive neurones in mice. T-MrVIa transgenic mice show a 44 +/- 7% reduction of tetrodotoxin-resistant (TTX-R) VGSC current densities. This inhibition is permanent, reversible and does not result in functional upregulation of TTX-sensitive (TTX-S) VGSCs, voltage-gated calcium channels (VGCCs) or transient receptor potential (TRP) channels present in nociceptive neurones. As a consequence of the reduction of TTX-R VGSC currents, t-MrVIa transgenic mice display decreased inflammatory mechanical hypersensitivity, cold pain insensitivity and reduced firing of cutaneous C-fibres sensitive to noxious cold temperatures. These data validate the use of genetically encoded t-toxins as a powerful tool to manipulate VGSCs in specific cell types within the mammalian nervous system. This novel genetic methodology can be used for circuit mapping and has the key advantage that it enables the dissection of the contribution of specific ionic currents to neuronal function and to behaviour.
Assuntos
Conotoxinas/farmacologia , Nociceptores/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Southern Blotting , Cromossomos Artificiais Bacterianos/genética , Conotoxinas/química , DNA/biossíntese , DNA/genética , Eletrofisiologia , Feminino , Imuno-Histoquímica , Hibridização In Situ , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Camundongos , Camundongos Transgênicos , Neurônios Aferentes/efeitos dos fármacos , Nociceptores/fisiologia , Oócitos/fisiologia , Dor/psicologia , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/inervação , Bloqueadores dos Canais de Sódio/química , Canais de Sódio/genética , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Xenopus laevisRESUMO
Laboratory male mice are often housed individually due to aggressive behavior or experimental requirements, though social isolation can cause welfare issues. As a strategy to refine housing of male mice, we introduce the separated pair housing system. A perforated transparent wall divides the cage into two compartments and allows olfactory, acoustic, and visual communication between the two mice but prevents fighting and injuries. Long-term effects of separated pair housing on well-being and distress of adult male C57BL/6JRj mice were investigated and compared with both single- and group-housed mice. Behavioral analysis after eight weeks in three different housing systems revealed no differences in burrowing performance, social interaction, anxiety, and stress hormone concentrations. However, pair-housed mice built more complex nests compared to single-housed mice and the nest position suggested that pair-housed mice preferred the close proximity to their cage mates. Moreover, pair-housed mice showed less locomotor activity compared to group- and single-housed mice. Body weight was higher in group-housed mice. All in all, no unambiguous long-term beneficial effects of pair housing on the well-being were found. However, the findings emphasized that effects of the housing systems on behavioral, physical, and biochemical parameters must be considered in the design of animal experimental studies.
Assuntos
Abrigo para Animais , Camundongos Endogâmicos C57BL/psicologia , Bem-Estar do Animal , Animais , Comportamento Animal , Corticosterona/análise , Combinação de Medicamentos , Compostos Ferrosos/química , Cabelo/química , Masculino , Camundongos , Mucinas/química , Interação Social , Testosterona/análise , Testosterona/metabolismoRESUMO
Lack of expression of neurofibromin in neurofibromatosis 1 and its lethal derivative, malignant peripheral nerve sheath tumors (MPNSTs), is thought to result in the overactivation of the Ras signaling pathway. Our previous studies have shown that cells with overactivation in the Ras pathway are more permissive to infection with herpes simplex virus 1 and its mutant version R3616. In this study, we show that among five different mouse MPNST cell lines, only the ones with elevated levels of Ras signaling are highly permissive to infection with oncolytic herpes G207. Specific inhibitors of the Ras, ERK, and JNK pathways all reduced the synthesis of viral proteins in MPNST cells. The cell lines that contained lower levels of Ras and decreased activation of downstream signaling components underwent an enhancement in apoptosis upon exposure to G207. Additionally, mouse SW10 Schwann cells were able to become infected by parental herpes but were found to be resistant to G207. The immortalization of these cell lines with the expression of SV40 large T antigen increased the levels of Ras activation and permissiveness to oncolytic herpes. A Ras/Raf kinase inhibitor reduced the synthesis of both herpes simplex virus-1 and G207 proteins in SW10 cells. The results of this study, therefore, introduce Ras signaling as a divergent turning point for the response of MPNST cells to an assault by oncolytic herpes.
Assuntos
Herpesvirus Humano 1/metabolismo , Neoplasias de Bainha Neural , Vírus Oncolíticos/metabolismo , Transdução de Sinais/fisiologia , Proteínas ras/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Herpesvirus Humano 1/genética , Humanos , Camundongos , Invasividade Neoplásica , Neoplasias de Bainha Neural/metabolismo , Neoplasias de Bainha Neural/patologia , Vírus Oncolíticos/genética , Replicação Viral , Proteínas ras/genéticaRESUMO
Pathological accumulation of misfolded α-synuclein (α-syn) in the brain plays a key role in the pathogenesis of Parkinson's disease, leading to neuronal dysfunction and motor disorders. The underlying mechanisms linking α-syn aggregations with neurotransmitter disturbance in Parkinson's brains are not well characterized. In the present study, we investigated transgenic mice expressing an aggregation-prone form of full-length human α-syn (h-α-synL62) linked to a signal sequence. These mice display dopamine depletion and progressive motor deficits. We detected accumulation of α-syn in cholinergic interneurons where they are colocalized with choline acetyltransferase. Using microdialysis, we measured acetylcholine levels in the striatum at baseline and during stimulation in the open field and with scopolamine. While no difference between wild-type and transgenic mice was detected in 3 month old mice, striatal acetylcholine levels at 9 months of age were significantly higher in transgenic mice. Concomitantly, high-affinity choline uptake was also increased while choline acetyltransferase and acetylcholine esterase activities were unchanged. The results suggest a disinhibition of acetylcholine release in α-syn transgenic mice.
Assuntos
Acetilcolina/metabolismo , Colina O-Acetiltransferase/metabolismo , Colina/metabolismo , Neurônios Colinérgicos/metabolismo , Corpo Estriado/metabolismo , alfa-Sinucleína/metabolismo , Acetilcolina/genética , Animais , Colina/genética , Colina O-Acetiltransferase/genética , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Microdiálise/métodos , alfa-Sinucleína/genéticaRESUMO
Malignant peripheral nerve sheath tumors (MPNST) are highly invasive soft tissue sarcomas that arise within the peripheral nerve and frequently metastasize. To identify molecular events contributing to malignant transformation in peripheral nerve, we compared eight cell lines derived from MPNSTs and seven normal human Schwann cell samples. We found that MPNST lines are heterogeneous in their in vitro growth rates and exhibit diverse alterations in expression of pRb, p53, p14(Arf), and p16(INK4a) proteins. All MPNST cell lines express the epidermal growth factor receptor and lack S100beta protein. Global gene expression profiling using Affymetrix oligonucleotide microarrays identified a 159-gene molecular signature distinguishing MPNST cell lines from normal Schwann cells, which was validated in Affymetrix microarray data generated from 45 primary MPNSTs. Expression of Schwann cell differentiation markers (SOX10, CNP, PMP22, and NGFR) was down-regulated in MPNSTs whereas neural crest stem cell markers, SOX9 and TWIST1, were overexpressed in MPNSTs. Previous studies have implicated TWIST1 in apoptosis inhibition, resistance to chemotherapy, and metastasis. Reducing TWIST1 expression in MPNST cells using small interfering RNA did not affect apoptosis or chemoresistance but inhibited cell chemotaxis. Our results highlight the use of gene expression profiling in identifying genes and molecular pathways that are potential biomarkers and/or therapeutic targets for treatment of MPNST and support the use of the MPNST cell lines as a primary analytic tool.
Assuntos
Neoplasias de Bainha Neural/genética , Células de Schwann/fisiologia , Apoptose/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica , Humanos , Neoplasias de Bainha Neural/metabolismo , Neoplasias de Bainha Neural/patologia , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/genética , Células de Schwann/metabolismo , Transfecção , Proteína 1 Relacionada a Twist/biossíntese , Proteína 1 Relacionada a Twist/genéticaRESUMO
Alpha-Synuclein (α-Syn) accumulation is considered a major risk factor for the development of synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies. We have generated mice overexpressing full-length human α-Syn fused to a membrane-targeting signal sequence under the control of the mouse Thy1-promotor. Three separate lines (L56, L58 and L62) with similar gene expression levels, but considerably heightened protein accumulation in L58 and L62, were established. In L62, there was widespread labelling of α-Syn immunoreactivity in brain including spinal cord, basal forebrain, cortex and striatum. Interestingly, there was no detectable α-Syn expression in dopaminergic neurones of the substantia nigra, but strong human α-Syn reactivity in glutamatergic synapses. The human α-Syn accumulated during aging and formed PK-resistant, thioflavin-binding aggregates. Mice displayed early onset bradykinesia and age progressive motor deficits. Functional alterations within the striatum were confirmed: L62 showed normal basal dopamine levels, but impaired dopamine release (upon amphetamine challenge) in the dorsal striatum measured by in vivo brain dialysis at 9 months of age. This impairment was coincident with a reduced response to amphetamine in the activity test. L62 further displayed greater sensitivity to low doses of the dopamine receptor 1 (D1) agonist SKF81297 but reacted normally to the D2 agonist quinpirole in the open field. Since accumulation of α-Syn aggregates in neurones and synapses and alterations in the dopaminergic tone are characteristics of PD, phenotypes reported for L62 present a good opportunity to further our understanding of motor dysfunction in PD and Lewy body dementia.
Assuntos
Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/genética , Animais , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Camundongos Transgênicos , Doença de Parkinson/genética , Fenótipo , Substância Negra/metabolismo , alfa-Sinucleína/metabolismoRESUMO
α-Synuclein (α-Syn) aggregation is a pathological feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease (PD). We have tested whether N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diaminium bis(hydromethanesulfonate) (leuco-methylthioninium bis(hydromethanesulfonate); LMTM), a tau aggregation inhibitor, affects α-Syn aggregation in vitro and in vivo. Both cellular and transgenic models in which the expression of full-length human α-Syn (h-α-Syn) fused with a signal sequence peptide to promote α-Syn aggregation were used. Aggregated α-Syn was observed following differentiation of N1E-115 neuroblastoma cells transfected with h-α-Syn. The appearance of aggregated α-Syn was inhibited by LMTM, with an EC50 of 1.1 µM, with minimal effect on h-α-Syn mRNA levels being observed. Two independent lines of mice (L58 and L62) transgenic for the same fusion protein accumulated neuronal h-α-Syn that, with aging, developed into fibrillary inclusions characterized by both resistance to proteinase K (PK)-cleavage and their ability to bind thiazin red. There was a significant decrease in α-Syn-positive neurons in multiple brain regions following oral treatment of male and female mice with LMTM administered daily for 6 weeks at 5 and 15 mg MT/kg. The early aggregates of α-Syn and the late-stage fibrillar inclusions were both susceptible to inhibition by LMTM, a treatment that also resulted in the rescue of movement and anxiety-related traits in these mice. The results suggest that LMTM may provide a potential disease modification therapy in PD and other synucleinopathies through the inhibition of α-Syn aggregation.
RESUMO
Neurofibromatosis type 1 (NF1) is a common genetic disorder of the nervous system resulting in neurofibromas and malignant peripheral nerve sheath tumors (MPNST). In this study, we report the modulation of murine and human MPNST cell growth by the fatty acids docosahexaenoic acid (DHA) and arachidonic acid (AA). DHA demonstrated a tendency to stimulate cell growth at low doses and induce apoptosis at high doses, paralleled by the activation of ERK and caspase-3. Furthermore, high-dose DHA reversed the stimulation of MPNST cell growth by a number of growth factors suggested to have a pathogenic effect in NF1 and inhibited MPNST growth in vivo. AA was found to have a reciprocal activity in vitro, stimulating MPNST cell growth at comparable concentrations and reducing DHA activation of ERK. These findings introduce fatty acids as a possible regulator of MPNST development in NF1 patients.
Assuntos
Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-6/farmacologia , Neoplasias de Bainha Neural/patologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Imuno-Histoquímica , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias de Bainha Neural/enzimologia , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PAK1, a Rac/CDC42-dependent Ser/Thr kinase, is required for the malignant growth of RAS transformants as well as both NF1-deficient and NF2-deficient cancer cells. FK228, a histone deacetylase (HDAC) inhibitor, suppresses the growth of more than 70% of human cancers in vivo including RAS transformants, breast cancers and prostate cancers by activating a set of genes including the tumor suppressors gelsolin and p21(WAF1), that block upstream and downstream of PAK1, respectively. Here we demonstrate that (1) the anti-PAK1 drug FK228 (0.1 nM) completely blocks the growth of both NF1-deficient and NF2-deficient cancer cells in vitro, and that (2) FK228 (2.5 mg/kg, i.p., twice a week) causes the complete regression of an NF1-deficient human malignant peripheral nerve sheath tumor (MPNST) xenograft in nude mice. This is the very first case where a chemical drug in clinical trials for cancers has ever worked so effectively on neurofibromatosis (experimental neurofibromas) in vivo.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Depsipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neurofibromina 1/deficiência , Transplante Heterólogo , Animais , Antibióticos Antineoplásicos/uso terapêutico , Depsipeptídeos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Neurofibromina 2/deficiência , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: A hallmark of rheumatoid arthritis (RA) is the chronic pain that accompanies inflammation and joint deformation. Patients with RA rate pain relief as the highest priority; however, few studies have addressed the efficacy and safety of therapies directed specifically toward pain pathways. The ω-conotoxin MVIIA (ziconotide) is used in humans to alleviate persistent pain syndromes, because it specifically blocks the voltage-gated calcium 2.2 (CaV 2.2) channel, which mediates the release of neurotransmitters and proinflammatory mediators from peripheral nociceptor nerve terminals. The aims of this study were to investigate whether blockade of CaV 2.2 can suppress arthritis pain, and to examine the progression of induced arthritis during persistent CaV 2.2 blockade. METHODS: Transgenic mice expressing a membrane-tethered form of MVIIA under the control of a nociceptor-specific gene (MVIIA-transgenic mice) were used in the experiments. The mice were subjected to unilateral induction of joint inflammation using a combination of antigen and collagen. RESULTS: CaV 2.2 blockade mediated by tethered MVIIA effectively suppressed arthritis-induced pain; however, in contrast to their wild-type littermates, which ultimately regained use of their injured joint as inflammation subsided, MVIIA-transgenic mice showed continued inflammation, with up-regulation of the osteoclast activator RANKL and concomitant joint and bone destruction. CONCLUSION: Taken together, our results indicate that alleviation of peripheral pain by blockade of CaV 2.2- mediated calcium influx and signaling in nociceptor sensory neurons impairs recovery from induced arthritis and point to the potentially devastating effects of using CaV 2.2 channel blockers as analgesics during inflammation.
Assuntos
Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Canais de Cálcio Tipo N/metabolismo , Dor Nociceptiva/metabolismo , Nociceptores/metabolismo , Ligante RANK/metabolismo , Joelho de Quadrúpedes/metabolismo , ômega-Conotoxinas/genética , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo N/imunologia , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Dor Nociceptiva/tratamento farmacológico , Dor Nociceptiva/imunologia , Joelho de Quadrúpedes/patologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima , ômega-Conotoxinas/uso terapêuticoRESUMO
A great deal of interest has been focused recently on the habenula and its critical role in aversion, negative-reward and drug dependence. Using a conditional mouse model of the ACh-synthesizing enzyme choline acetyltransferase (Chat), we report that local elimination of acetylcholine (ACh) in medial habenula (MHb) neurons alters glutamate corelease and presynaptic facilitation. Electron microscopy and immuno-isolation analyses revealed colocalization of ACh and glutamate vesicular transporters in synaptic vesicles (SVs) in the central IPN. Glutamate reuptake in SVs prepared from the IPN was increased by ACh, indicating vesicular synergy. Mice lacking CHAT in habenular neurons were insensitive to nicotine-conditioned reward and withdrawal. These data demonstrate that ACh controls the quantal size and release frequency of glutamate at habenular synapses, and suggest that the synergistic functions of ACh and glutamate may be generally important for modulation of cholinergic circuit function and behavior.
Assuntos
Acetilcolina/metabolismo , Neurônios Colinérgicos/fisiologia , Ácido Glutâmico/metabolismo , Habenula/fisiologia , Sinapses/efeitos dos fármacos , Tabagismo , Animais , Condicionamento Clássico , CamundongosRESUMO
Nerve sheath tumors are the most common tumors of Neurofibromatosis type 1 (NF1) patients. Dermal neurofibromas develop in nearly all NF1-patients, whereas plexiform neurofibromas are only observed in one-third of the patients. NF1-patients have about a 10% lifetime risk for developing malignant pheripheral nerve sheath tumors (MPNST). The origin of these tumors is thought to be the Schwann cell lacking functional neurofibromin. However, additional genetic alterations are likely to modulate tumor biology and to contribute to individual nerve sheath tumor entities. To gain insight into the molecular events and to determine whether these tumors can be classified according to gene expression profiles, we performed expression analysis applying cDNA array technology. Nine dermal neurofibromas, 7 plexiform neurofibromas, ten MPNST and two MPNST cell cultures were examined. All tumors but 6 sporadic MPNST were obtained from NF1-patients. We detected significant differences in gene expression patterns between neurofibromas and MPNST and between dermal neurofibromas and plexiform neurofibromas. Tumor class prediction agreed in all but one case with histological and clinical classification. NF1-associated and sporadic MPNST could not be distinguished by their gene expression patterns. We present a panel of discriminating genes that may assist subclassification of nerve sheath tumors.
Assuntos
Neoplasias de Bainha Neural/classificação , Neoplasias de Bainha Neural/genética , Neurofibroma/classificação , Neurofibroma/genética , Neurofibromatose 1/genética , Adolescente , Adulto , Idoso , Linhagem Celular Tumoral , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador , Hibridização In Situ , Pessoa de Meia-Idade , Neurofibroma Plexiforme/classificação , Neurofibroma Plexiforme/genética , Análise de Sequência com Séries de OligonucleotídeosRESUMO
BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) are neoplasms leading to death in most cases. Patients with Neurofibromatosis type 1 have an increased risk of developing this malignancy. The metabolites of the inactive prodrug Sulindac, Sulindac Sulfide and Sulindac Sulfone (Exisulind) are new chemopreventive agents that show promising results in the treatment of different cancer types. In this study we examined the antineoplastic effect of these compounds on primary cells derived from two MPNSTs of Neurofibromatosis type 1 patients. RESULTS: Exisulind and Sulindac Sulfide showed a dramatic time- and dose-dependent growth inhibitory effect with IC50-values of 120 microM and 63 microM, respectively. The decrease in viability of the tested cells correlated with induction of apoptosis. Treatment with 500 microM Exisulind and 125 microM Sulindac Sulfide for a period of 2 days increased the rate of apoptosis 21-27-fold compared to untreated cells. Reduced expression of RAS-GTP and phosphorylated ERK1/2 was detected in treated MPNST cells. Moreover, elevated levels of phosphorylated SAPK/JNK were found after drug treatment, and low activation of cleaved caspase-3 was seen. CONCLUSIONS: Our results suggest that this class of compounds may be of therapeutic benefit for Neurofibromatosis type 1 patients with MPNST.
RESUMO
The CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the α5, α3, and ß4 nicotinic acetylcholine receptor (nAChR) subunits, has been linked to nicotine dependence. The habenulo-interpeduncular (Hb-IPN) tract is particularly enriched in α3ß4 nAChRs. We recently showed that modulation of these receptors in the medial habenula (MHb) in mice altered nicotine consumption. Given that ß4 is rate-limiting for receptor activity and that single nucleotide polymorphisms (SNPs) in CHRNB4 have been linked to altered risk of nicotine dependence in humans, we were interested in determining the contribution of allelic variants of ß4 to nicotine receptor activity in the MHb. We screened for missense SNPs that had allele frequencies >0.0005 and introduced the corresponding substitutions in Chrnb4. Fourteen variants were analyzed by co-expression with α3. We found that ß4A90I and ß4T374I variants, previously shown to associate with reduced risk of smoking, and an additional variant ß4D447Y, significantly increased nicotine-evoked current amplitudes, while ß4R348C, the mutation most frequently encountered in sporadic amyotrophic lateral sclerosis (sALS), showed reduced nicotine currents. We employed lentiviruses to express ß4 or ß4 variants in the MHb. Immunoprecipitation studies confirmed that ß4 lentiviral-mediated expression leads to specific upregulation of α3ß4 but not ß2 nAChRs in the Mhb. Mice injected with the ß4-containing virus showed pronounced aversion to nicotine as previously observed in transgenic Tabac mice overexpressing Chrnb4 at endogenous sites including the MHb. Habenular expression of the ß4 gain-of-function allele T374I also resulted in strong aversion, while transduction with the ß4 loss-of function allele R348C failed to induce nicotine aversion. Altogether, these data confirm the critical role of habenular ß4 in nicotine consumption, and identify specific SNPs in CHRNB4 that modify nicotine-elicited currents and alter nicotine consumption in mice.